Scientific understanding of the functional role of post-transcriptionally modified nucleosides in ribonucleic acids (RNAs) is limited in large part by the lack of methods that can routinely identify and map modifications onto a primary RNA sequence. The long-term goal of this research continues to be to develop appropriate mass spectrometric approaches that enable biological studies into the functional significance of modified RNAs. This renewal is focused on the specific goal of improving RNA modification mapping by mass spectrometry. The existing protocol used in the discipline was developed over 30 years ago and has significant limitations for researchers seeking to discover new modifications within complex mixtures of cellular RNAs. This renewal is separated into three aims that will result in the creation of a modification mapping protocol that can enable modern biological studies into RNA modification patterns, including the growing area of RNA epigenetics. The first aim will enable discovery-based RNA modification mapping by making available three new base-specific endonucleases. This aim ensures appropriate sequence coverage in modification mapping experiments. The next two aims focus on improving detection and sequencing of modified RNA digestion products by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The second aim revises the strategy used for LC-MS/MS identification of modified nucleosides within larger RNAs. By eliminating wasteful LC-MS/MS sequencing of unmodified regions of any RNA, the resulting MS/MS data will be enriched in information about the mass and sequence location of any particular modification. The final aim will create a data independent acquisition (DIA)-based LC-MS/MS strategy for RNA modification mapping. This research plan will have significant impacts on multiple research fields. Discovery-based modification mapping can be used to expand our knowledge of eukaryotic RNA modification patterns, including modifications that are important for biological function. The new protocol will enable research into xenobiotic modifications to RNA including processes such as RNA oxidative damage, which may be significant in aging-related diseases such as cardiovascular disease, cancer and neurological impairment. Moreover, the bioanalytical developments to result from this research can be applied to scientific investigations that seek to understand how the cell regulates RNA modification patterns as well as how variable RNA modification patterns affect other cellular regulatory processes.